Vaccinia virus has a genome of about 185,000 base pairs that encodes approximately 200 polypeptides. These genes are expressed within the cytoplasm in a coordinated fashion, so that some polypeptides are made before, and others after, DNA replication. Enzymes and factors needed for early transcription are packaged within the infectious particle, while those needed for late transcription are present in the cytoplasm of infected cells. The aim of this project is to determine the mechanisms regulating gene expression. Progress in understanding early and late transcription has been made this past year. Several viral genes required for transcription were identified. These include an RNA polymerase subunit of 18,000 daltons, both subunits of the early transcription factor VETF, and three transactivators of late gene expression. Evidence was obtained for a cascade mechanism of gene regulation in which the early transcription factors are made late during the previous infection cycle and packaged in virions, the intermediate factors are made early in infection, and the late factors are made at intermediate times. The DNA present in the infecting virus particle serves as a template for transcription of early genes but naked or newly replicated DNA is required as a template for intermediate and late genes.